The present invention relates in general to compositions and methods of modulating the function of proteins involved in protein-protein interactions. It relates more specifically to modulating the function of a first protein of a pair of interacting proteins wherein a second protein of the pair contains a xe2x80x9cWD-40xe2x80x9d or xe2x80x9cxcex2-transducinxe2x80x9d amino acid repeat motif.
Many intracellular processes are carried out or regulated by multi-subunit protein complexes that become active or repressed by the association or dissociation of individual polypeptide subunits.
One such group or family of proteins is related to the xcex2 subunit of transducin. Members of this group are all at least somewhat homologous to the xcex2-subunit of transducin at the amino acid level, and contain a varying number of repeats of a particular motif identified in xcex2-transducin. The repeats have been termed xe2x80x9cxcex2-transducinxe2x80x9d, or xe2x80x9cWD-40xe2x80x9d repeats (Fong, et al.).
Among the members of this protein family (Duronio, et al.) are the Gxcex2 subunits that couple many receptors to their intracellular effector molecules, Gxcex2/xcex3 subunits that anchor another protein kinase (the xcex2-adrenergic receptor kinase, xcex2ARK), DNA binding proteins and yeast cell cycle proteins. All of these require a transient protein-protein interaction for their function. However, the sequences at the interface of these proteins and their partners have not been identified.
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The invention includes, in one aspect, a polypeptide composition effective to alter the activity of a first protein, such as protein kinase C, or xcex2-adrenergic receptor kinase (xcex2ARK). The polypeptide blocks or inhibits an interaction, such as a binding interaction, between the first protein and a second protein containing a WD-40 region.
The polypeptide contains between 4 and 50 amino acids whose sequence is the same as a sequence of the same length in the WD-40 region of the second protein.
The polypeptide may block the binding of the first to the second protein, or may be an agonist or antagonist of the first protein. The WD-40 region preferably has an amino acid sequence homologous or identical to the sequences defined by SEQ ID NO:76-261.
In a second embodiment, the invention includes a method of altering the activity of the first protein of the type defined above. The method includes selecting a polypeptide having between 4 and 50 amino acids whose sequence is the same as a sequence of the same length in the WD-40 region of the second protein, and contacting the polypeptide with the first protein under conditions which allow the formation of a complex between the polypeptide and the first protein, where this interaction alters the activity of the first protein.
In one embodiment, the contacting is effective to inhibit the interaction between the first and second proteins. In another embodiment, the contacting is effective to stimulate the activity of the first protein.
In still another embodiment, the contacting is effective to inhibit the activity of the first protein.
The polypeptide preferably has an amino acid sequence homologous or identical to the sequences defined by SEQ ID NO:76-261.
In a more specific aspect of the invention, the invention includes a polypeptide composition effective to alter the activity of protein kinase C, where the protein kinase C interacts with a second protein, and the second protein contains at least one WD-40 region. The polypeptide has between 4 and 50 amino acids whose sequence is the same as a sequence of the same length in the WD-40 region of the second protein.
In a preferred embodiment, the second protein is a receptor for activated protein kinase C, and has the sequence represented by SEQ ID NO:27.
In other specific embodiments, the polypeptide is (i) an agonist of protein kinase C, and the polypeptide has the sequence represented by SEQ ID NO:7; (ii) an antagonist of the activity of protein kinase C; and/or (iii) an inhibitor of the interaction between protein kinase C and the second protein. In the latter embodiment, the polypeptide has sequence corresponding to SEQ ID NO:4 or SEQ ID NO:7.
The WD-40 region preferably has an amino acid sequence homologous or identical to SEQ ID NO:69-75.
In a related embodiment, the invention includes a method of altering the activity of a protein kinase C that interacts with a second protein, where said second protein contains at least one WD-40 region.
The method includes selecting a polypeptide having between 4 and 50 amino acids whose sequence is the same as a sequence of the same length in the WD-40 region of the second protein, and contacting the polypeptide with the protein kinase C under conditions which allow the formation of a complex between the polypeptide and the protein kinase C, where said interaction alters the activity of said protein kinase C.
Other aspects of the invention include the polypeptide compositions of the invention wherein said polypeptide is coupled to a solid support, as well as a method to bind selectively said first protein which method comprises contacting a sample putatively containing said first protein with the polypeptide composition bound to solid support and removing any unbound components of the sample from said composition.
In still another aspect, the invention relates to a method to assess the interaction of a first protein with a polypeptide represented by an amino acid sequence contained in a second protein, wherein said second protein contains at least one WD-40 region, which method comprises contacting a sample containing said first protein with a polypeptide composition wherein the polypeptide has between 4 and 50 amino acids whose sequence is the same as the sequence of the same length in the WD-40 region of the second protein, and observing any interaction of the first protein with said polypeptide composition. The invention also concerns a method to assess the ability of a candidate compound to bind a first protein which method comprises contacting said first protein with a polypeptide composition which binds said first protein, wherein the polypeptide of said composition has between 4 and 50 amino acids whose sequence is the same as a sequence of the same length in a WD-40 region of a second protein which interacts with said first protein, in the presence and absence of said candidate compound; and measuring the binding of said polypeptide in the presence and in the absence of said candidate, wherein decreased binding of the polypeptide in the presence as opposed to the absence of said candidate indicates that said candidate binds to said first protein.
In still another aspect, the invention is directed to recombinant materials for the production of the polypeptides of the invention and methods for their production.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.